Specificity and Actions of an Arylaspartate Inhibitor of Glutamate Transport at the Schaffer Collateral-CA1 Pyramidal Cell Synapse

Abstract

In this study we characterized the pharmacological selectivity and physiological actions of a new arylaspartate glutamate transporter blocker, L-threo-ß-benzylaspartate (L-TBA). At concentrations up to 100 µM, L-TBA did not act as an AMPA receptor (AMPAR) or NMDA receptor (NMDAR) agonist or antagonist when applied to outside-out patches from mouse hippocampal CA1 pyramidal neurons. L-TBA had no effect on the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded at the Schaffer collateral-CA1 pyramidal cell synapse. Excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons were unaffected by L-TBA in the presence of physiological extracellular Mg2+ concentrations, but in Mg2+-free solution, EPSCs were significantly prolonged as a consequence of increased NMDAR activity. Although L-TBA exhibited approximately four-fold selectivity for neuronal EAAT3 over glial EAAT1/EAAT2 transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg2+ was the same in hippocampal slices from EAAT3 +/+ and EAAT3 −/− mice, suggesting that TBA effects were primarily due to block of glial transporters. Consistent with this, L-TBA blocked synaptically evoked transporter currents in CA1 astrocytes with a potency in accord with its block of heterologously expressed glial transporters. Extracellular recording in the presence of physiological Mg2+ revealed that L-TBA prolonged fEPSPs in a frequency-dependent manner by selectively increasing the NMDAR-mediated component of the fEPSP during short bursts of activity. The data indicate that glial glutamate transporters play a dominant role in limiting extrasynaptic transmitter diffusion and binding to NMDARs. Furthermore, NMDAR signaling is primarily limited by voltage-dependent Mg2+ block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling.

Identifying neurotransmitter spill-over in hippocampal field recordings

“…We conclude that, consistent with the results in [32], non-local glial transporters are primarily responsible for limiting spill-over of synaptically released glutamate, and we establish mathematically that a model of co-localized transporters and receptors (as would be the case with neuronal transporters) cannot reproduce the paired pulse experimental results with L-TBA.…”

“…The action of neurotransmitter transporters is key in many of these studies, since the inhibition of these transporters can lead to excess neurotransmitter in the extracellular space. More recent studies of this phenomenon include Scimemi et al [30] and Sun et al [32]. An overview of glutamate spill-over and transporter action is given in Diamond [12].…”

“…The glutamate uptake blocker DL-TBOA, blocks both neuronal and glial transporters, and a newer transport blocker, L-threo-beta-benzylaspartate, L-TBA, exhibits a slight selectivity for EAAT3 over both EAAT1 and EAAT2. A recent paper by Sun et al [32], studies the characteristics of L-TBA in detail.…”

The Density of EAAC1 (EAAT3) Glutamate Transporters Expressed by Neurons in the Mammalian CNS

“…Some immunocytochemical studies conducted before EAAC1 knock-out mice became available noted labeling of GABAergic terminals (He et al, 2000(He et al, , 2001 in the deep cerebellar nuclei (Rothstein et al, 1994). A study based on antisense knockdown concluded that EAAC1 is important for GABA synthesis in GABAergic terminals (Sepkuty et al, 2002), but the antisense probes used may have had unrecognized effects because EAAC1 knockdown using the same method (Rothstein et al, 1996) lead to a larger reduction in glutamate uptake activity than can be attributed to this transporter (see data in the studies by Danbolt et al, 1992;Haugeto et al, 1996;Jahr, 1997, 1998;Peghini et al, 1997;Tanaka et al, 1997;Furness et al, 2008;Sun et al, 2011).…”

“…In contrast, the role of EAAC1 (EAAT3) is still debated. Immunoisolation of transport activity (Haugeto et al, 1996), deletion of the GLT-1 (slc1a2) gene Jahr, 1997, 1998;Tanaka et al, 1997;Sun et al, 2011), and the mild phenotype of EAAC1-deficient mice (Peghini et al, 1997) suggest that EAAC1-mediated glutamate uptake is negligible compared with that of GLT-1. However, EAAC1-deficient mice suffer from dicarboxylic aminoaciduria (Peghini et al, 1997) and premature aging (Chen and Swanson, 2003;Aoyama et al, 2006;Berman et al, 2011).…”